Note: While transfection conditions have been optimized to allow highly efficient delivery of Stealth™ RNAi into 3T3 L1 cells, other factors related to the target gene of interest including the transcription rate of the target gene, the stability of the resulting protein, and efficacy of the Stealth™ RNAi sequence chosen can influence the degree of target gene knockdown observed. Take these factors into consideration when designing your RNAi experiment.

Transfection Procedure

Use this procedure to transfect Stealth™ RNAi (or siRNA) into 3T3 L1 cells using Lipofectamine® 2000 in a 24-well format. For other formats, see the table in Recommended Reagent Amounts and Volumes for the appropriate reagent amounts to add.

Tip: To reduce well-to-well variability when transfecting multiple replicates (e.g. triplicates), proportionally scale up the reagent volumes to form complexes (Step 2), then aliquot an equal volume of complexes into each well.

One day before transfection, plate 3 x 104 3T3 L1 cells in 400 μl of growth medium without antibiotics per well. Cells should be 60-70% confluent at the time of transfection.

After the 15-minute incubation, combine the diluted Stealth™ RNAi and the diluted Lipofectamine® 2000 (total volume ~ 100 μl). Mix gently and incubate for 15 minutes at room temperature to allow complexes to form (solution may appear cloudy).

Add the ~100 μl of Stealth™ RNAi-Lipofectamine® 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.

Incubate the cells at 37°C in a humidified CO2 incubator for 16-24 hours as appropriate until you are ready to assay for gene knockdown. It is not necessary to remove the complexes or change the medium; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.

Recommended Reagent Amounts and Volumes

To transfect 3T3 L1 cells in different tissue culture formats, vary the amounts of Stealth™ RNAi (or siRNA), Lipofectamine® 2000, cells, and medium used in proportion to the relative surface area, as shown in the table. Note: 20 μM Stealth™ RNAi or siRNA = 20 pmol/μl.